Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity

Yuanmei Zhu; Danwei Yu; Hongxia Yan; Huihui Chong; Yuxian He

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.26.009233 doi: bioRxiv preprint 4 suggested that ACE2-binding affinity of the RBD of SARS-CoV-2 is up to 20-fold higher 55 than that of SARS-CoV, which may contribute to the significantly increased infectivity and 56 transmissibility (6). The receptor-binding deem to trigger large conformational changes in the 57 S complex, which destabilize the prefusion trimer resulting in shedding of the S1 subunit and 58 activate the fusogenic activity of the S2 subunit (9-11). As illustrated in Fig. 1 , the sequence 59 structure of S2 contains an N-terminal fusion peptide (FP), heptad repeat 1 (HR1), heptad 60 repeat 2 (HR2), transmembrane region (TM), and cytoplasmic tail (CT). During the fusion 61 process, the FP is exposed and inserts into the target cell membrane, leading S2 in a 62 prehairpin intermediate that bridges the viral and cell membranes; then, three HR1 segments 63 self-assemble a trimeric coiled-coil and three HR2 segments fold into the grooves on the 64 surface of the HR1 inner core, thereby resulting a six-helical bundle (6-HB) structure that 65 drives the two membranes in close apposition for fusion. SARS-CoV-2 infection. We found that different from that of SARS-CoV, the S protein of 80 SARS-CoV-2 has a high cell fusion activity; then, we designed and characterized several 86 In the earlier time point, we would like to experimentally verify whether SARS-CoV-2 uses 87 human ACE2 as a receptor for cell entry, thus we generated its S protein pseudotyped 88 lentiviral particles. The SARS-CoV and vesicular stomatitis virus (VSV-G) pseudoviruses 89 were also prepared for comparison. As shown in Fig. 2A , all of three pseudoviruses 90 efficiently infected 293T cells that stably overexpress ACE2 (293T/ACE2); however, the 91 infectivity SARS-CoV-2 and SARS-CoV dramatically decreased in 239T cells which express 92 a low level of endogenous ACE2. As a virus control, VSV-G pesudovirus entered 239T cells 93 even more efficiently relative its infectivity in 293T/ACE2 cells. 94 We further compared the fusion activity of viral S protein in 293T and 293T/ACE2 cells 95 by applying a DSP-based cell-cell fusion assay. As shown in Fig. 2B , both the S proteins of 96 SARS-CoV-2 and SARS-CoV displayed a weak fusion activity in 293T cells, but they 97 showed significantly increased capacities to mediate cell fusion with 293T/ACE2 cells. These 98 author/funder. All rights reserved. No reuse allowed without permission.